Somatostatin Ligands Research Articles

Growth hormone increase by luteinizing hormone-releasing hormone reflects gonadotroph-related characteristics in acromegaly

PurposeWe previously showed the clinical characteristics of acromegaly with a paradoxical growth hormone (GH) response to oral glucose or thyrotropin-releasing hormone. However, the clinical characteristics of acromegaly with an increased GH response to luteinizing hormone-releasing hormone (LHRH responders) remain unclear. The aim of the present study was to evaluate the clinical characteristics, especially gonadotroph-related characteristics of LHRH responders in acromegaly.MethodsThe clinical characteristics of 33 LHRH responders and 81 LHRH nonresponders were compared.ResultsNo differences in age, sex or basal serum levels of GH, insulin-like growth factor-1 (IGF-1), and gonadotropin were observed between the two groups. Steroidogenic factor 1 (SF-1), gonadotropin-releasing hormone receptor (GnRHR), and LH expression was more frequently observed in LHRH responders (P < 0.05). In addition, a greater increased rate of GH after LHRH loading, and the proportion of GnRHR and gonadotropin expression was observed in pituitary tumor with SF-1 expression than that without the expression (P < 0.01). LHRH responders showed a greater GH decrease in the octreotide test and a greater IGF-1 decrease after first-generation somatostatin ligand than LHRH nonresponders (P < 0.05). Furthermore, the proportion of hypointense pituitary tumors on T2-weighted magnetic resonance imaging and tumors with densely granulated type was higher in LHRH responders than in LHRH nonresponders, respectively (P < 0.05). No difference between the two groups was observed in either somatostatin receptor 2 or 5 expression.ConclusionsThe increased GH response to LHRH is associated with the gonadotroph-related characteristics. This response may reflect the biological characteristics of somatotroph tumors.

A novel somatostatin receptor ligand for human ACTH - and GH -secreting pituitary adenomas.

Somatostatin receptor ligands have come to play a pivotal role in the treatment of both ACTH- and GH-secreting pituitary adenomas. Clinical efficacy averages 30-50%, thus a considerable number of patients with Cushing's disease or acromegaly remain unresponsive to this therapeutic approach. HTL0030310 is a new somatostatin receptor ligand selective for subtype 5 over subtype 2, thus with a different receptor profile compared to clinical somatostatin receptor ligands. Assessment of the effect of HTL0030310 on hormone secretion in human ACTH- and GH-secreting pituitary adenomas in vitro. Primary cultures from 3 ACTH-secreting and 5 GH-secreting pituitary adenomas were treated with 1, 10 and 100 nM HTL0030310 alone or with 10 nM CRH or GHRH, respectively. Parallel incubations with 10 nM pasireotide were also carried out. ACTH and GH secretion were assessed after 4 and 24 hour incubation; SSTR2, SSTR3, SSTR5, GH and POMC expression were evaluated after 24 hours. HTL0030310 reduced unchallenged ACTH and POMC levels up to 50% in 2 ACTH-secreting adenomas and blunted CRH-stimulated ACTH/POMC by 20-70% in all 3 specimens. A reduction in spontaneous GH secretion was observed in 4 GH-secreting adenomas and in 2 specimens during GHRH co-incubation. SSTRs expression was detected in all specimens. This first study on a novel somatostatin receptor 5-preferring ligand indicates that HTL0030310 can inhibit hormonal secretion in human ACTH- and GH-secreting pituitary adenomas. These findings suggest a potential new avenue for somatostatin ligands in the treatment of Cushing's disease and acromegaly.

THU050 The Role Of The GH Receptor Polymorphism As Prognostic Factor Of Vertebral Fractures In Acromegaly Patients Resistant To First Generation SSAs And Treated With GH Receptor Antagonist Or Second Generation Somatostatin Ligand

Abstract Disclosure: F. Costanza: None. S. Chiloiro: None. A. Giampietro: None. P. Mattogno: None. A. Infante: None. F. Angelini: None. L. Lauretti: None. A. Olivi: None. A. Pontecorvi: None. F. Doglietto: None. L. De Marinis: None. A. Bianchi: None. Acromegaly is associated with skeletal fragility and an increased prevalence of vertebral fractures (VF). In recent years several authors have tried to investigate the markers that can predict the risk of bone fragility in this endocrine disorder. Two different isoforms of the GH receptor (GHR) have been described so far, which differ in the presence or absence of a transcript of exon 3 of the GHR gene. Both isoforms produce a functional receptor, but the exon 3-deleted isoforms (d3-GHR) have greater sensitivity to endogenous and recombinant GH than the full-length isoform (fl-GHR). We conducted a longitudinal, retrospective, observational, single-center study of first-generation SSA-resistant acromegaly patients, with the aim of investigating the role of GHR polymorphism as a prognostic factor of incidental VF (I-VF) in first-generation SSA-resistant acromegalic patients and treated with GH receptor antagonist (Pegvisomant) or second-generation somatostatin ligand (Pasireotide Lar).72 patients with acromegaly were included. 28 patients carried d3-GHR isoform (38.9%) and 44 patients carried fl-GHR isoform (61.1%). At baseline, all patients were affected by active acromegaly; 46 patients were treated with Pegvisomant in combination with first generation SSA and 26 were treated with Pasireotide Lar. 18 patients (25%) carried P-VFs. At last follow-up, 58 patients achieved biochemical control of acromegaly (80.6%). 14 patients experienced the occurrence of I-VFs. From the group treated with Pegvisomant in combination with first generation SSA, 32 patients carried fl-GHR polymorphism and 14 carried d3-GHR polymorphism. At baseline, 10 patients (21.7%) were I-VF carriers. At follow-up, 8 patients developed P-VF (17.4%). I-VF occurred more frequently in patients carrying fl-GHR isoform compared to d3-GHR (p=0.04). From the group treated with Pasireotide Lar, 12 patients carried fl-GHR isoform and 14 patients carried d3-GHR isoform. At baseline, 8 patients (30.8%) were I-VF carriers. At follow-up, 6 patients developed P-VF (23.1%). I-VF occurred more frequently in patients carrying d3-GHR isoform than fl-GHR (p=0.01) and in patients with P-VF as compared to patients without P-VF (p=0.05).In conclusion, the GH receptor polymorphism could assume greater relevance as a prognostic factor of VF in acromegaly patients. In the future, the knowledge of the GHR polymorphism may improve the therapeutic approach of acromegaly patients, tailored to the individual patient, in the context of personalized medicine. Presentation: Thursday, June 15, 2023

THU055 The Acro-TIME Score: A New Clinical, Pathological And Immune Integrative Approach To Early Identify Acromegaly Patients Resistant To Treatment With First Generation Somatostatin Ligands

Abstract Disclosure: S. Chiloiro: None. A. Giampietro: None. M. Gessi: None. L. Lauretti: None. M. Pier Paolo: None. A. Carlino: None. A. Olivi: None. G. RIndi: None. A. Pontecorvi: None. L. De Marinis: None. F. Doglietto: None. A. Bianchi: None. Introduction: Somatotropinomas are benign neoplasia, with a heterogenous clinical behavior. The acromegaly control is reached in 25%-65% of patients (pts) treated with first generation somatostatin ligands (fg-SRLs). Tumor microenvironment reflects the interaction between tumor cells and the host immune system, potentially regulating tumor behavior and therapy outcome. We aim to develop a score that includes clinical, pathological and immune markers to early identify fg-SRLs resistant acromegaly pts, that require second line treatments. Patients: 43 consecutive acromegaly pts were included according the following criteria (1) first line treatment with surgery, (2) post-surgical fg-SRLs therapy (3) availability of tumor samples for experiments. Pts not-naïve to acromegaly therapies before surgery, with history of radiotherapy of head and neck within 10 years before pituitary surgery, with immune-related disease were ruled out. Results: Eighteen pts (41.9%) were fg-SRLs resistant: 14 were females (77.8%), with a median age of 36.5 (IQR:13) and with cavernous sinus invasion in 12 cases (66.7%). At histological examination, Ki-67 was &amp;lt;1.5% in 17 cases (39.5%). The SSTR2A Volante scores were 0-1 in 5 cases (11.6%) and 2-3 in 38 cases (88.4%). Tumor-infiltering CD4+ lymphocytes was 4.9/HFP (IQR: 8), CD8+ lymphocytes was 11/HFP (IQR:14) and CD68+ cells was 60/HFP (IQR:69). The ratio CD68+/CD8+ cells was 5.2 (IQR: 5). We analysed 18 clinical, pathological and immune features as possible predictors of fg-SRLs response. Fg-SRLs resistance was associated to age at acromegaly diagnosis &amp;lt;37 years (AUC: 0.72 OR: 2 95%IC: 1.1-4 p=0.04), cavernous sinus invasion (OR: 9.3 95%IC:1.4-61 p&amp;lt;0.001), Ki-67&amp;gt;1.5% (OR: 3.2 95%IC:1.2-13.1 p=0.04), score 0-1 of SSTR2A (OR: 2.7 95%IC: 1.7-4.1 p=0.03), ratio CD68+/CD8+ cells&amp;lt;5.7/HPF (AUC: 0.709 OR: 4.9 95%IC:1.2-19.2 p=0.03) and persistence of post-surgery residual tumor (OR: 2.5 95%IC:1.3-4.7 p=0.004). These variables were analysed in a logistic regression model, yielding a beta coefficient of 3.7 for age &amp;gt;37 years; of -3 for cavernous sinus invasion; of -0.2 for Ki-67&amp;gt;1.5%; of 20 for SSTR2A score 2-3; of -0.9 for CD68+/CD8+cells ratio &amp;gt;5.7/HFP; and of -0.9 for persistence of post-surgery residual. We assigned a score to each covariate proportional to its beta coefficient, yielding a cumulative score for each pts. The score values ranged from 18.5 to 24 in cases responsive to fg-SRLS and from -5.5 to 21.5 in fg-SRLs resistant cases. A score &amp;lt;19 was chosen as cut-point to identify fg-SRLs resistance (AUC: 0.059 p&amp;lt;0.001 95%IC: 0.0-0.126). A score &amp;lt;19 was associated to fg-SRLs resistance in 84.6% of cases (p&amp;lt;0.001 OR: 3.7 95%IC:1.7-6.7). Conclusions: This new score integrates clinical, pathological, immunological data and may predict fg-SRLs resistance and the need of second line therapies. This study was supported by 2022 Arrigo Recordati International Prize Presentation: Thursday, June 15, 2023

The Role of the GH Receptor Polymorphism as Prognostic Factor of Vertebral Fractures in Acromegaly Patients Resistant to First Generation SSAs and Treated with GH Receptor Antagonist or Second-Generation Somatostatin Ligand

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

The acro-TIME score: a new clinical, pathological and immune integrative approach to early identify acromegaly patients resistant to treatment with first generation somatostatin ligands

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Reduced irisin levels in patients with acromegaly.

Several metabolic disturbances are seen in acromegaly however, data regarding the contribution of irisin to these disturbances is currently insufficient. In a cohort of patients with acromegaly, we measured serum irisin levels in active and controlled cases and determined independent factors that effect serum irisin including fibronectin type III domain-containing protein 5 (FNDC5) genotyping. A cross-sectional case-control study including 46 patients with acromegaly (28F/18M, age: 50.3±12.1year, BMI: 30.7±5.1kg/m2) and 81 age-, gender-, body mass index- and body composition-matched healthy controls was conducted. 15 acromegalic patients (33%) had active disease. Irisin levels were measured by enzyme-linked immunosorbent assay. Three different regions (rs3480, rs1746661, and rs16835198) of FNDC5 were subjected to polymorphism analyses. Both groups were overweight and had similar body composition. Irisin levels were lower in patients with acromegaly than controls (median [IQR]: 44.8 [41.7-46.7] ng/mL vs. 51.7 [45.5-60.1] ng/mL, p≤0.001, respectively). Active and controlled patients had similar irisin levels. Irisin was not correlated with growth hormone (GH), insulin-like growth factor 1 (IGF-1), and IGF-1 index. In multiple linear regression model, somatostatin receptor ligand use (β=-20.30, 95% CI [-34]-[-6], p=0.006) was determined as the only independent factor that affect serum irisin. Serum irisin levels are low in patients with acromegaly who are on somatostatin receptor ligand therapy. Single nucleotide polymorphisms (SNPs) of FNDC5 have no independent effects on circulating irisin levels under somatostatin ligand action. Endocrine muscle functions also seem to be regulated by somatostatin action, which requires further studies.

First-generation somatostatin ligand receptor treatment in a pregnant patient with a neuroendocrine tumor with liver metastases.

SummaryThis rare case describes the course of a pregnancy in a patient with a disseminated small intestinal neuroendocrine tumor. The patient received treatment with first-generation somatostatin ligand receptor (SLR) every 4 weeks and had stable disease for several years before her pregnancy. First-generation SLR treatment was initially paused after detection of the pregnancy. During pregnancy, the patient experienced moderate gastro-intestinal discomfort and fatigue, which was considered predominantly pregnancy related. However, since symptoms could be linked to the patient’s cancer, treatment was resumed after the first trimester. Chromogranin-A measurements remained stable throughout pregnancy and was paralleled by the absence of diarrhea and only minor flushing. She gave birth by elective caesarean section in week 37 to a healthy baby. Subsequent follow up imaging immediately after and 10 months postpartum showed no disease progression. The safety profile of SLR treatment during pregnancy in the context of disseminated neuroendocrine tumors (NET) is discussed.Learning pointsNeuroendocrine neoplasms (NEN) are rare cancers often occurring in the gastro-intestinal tract or lungs.Many patients with NEN live for several years with disseminated disease.SLR treatment has been given to pregnant patients before; often patients with acromegaly. Pregnancies are reported uneventful.This patient completed an uneventful pregnancy while receiving SLR treatment for disseminated neuroendocrine disease and gave birth to a healthy baby.More research regarding long term effects and safety signals of SLR treatment during pregnancy are much needed.

In Vivo Instability of 177Lu-DOTATATE During Peptide Receptor Radionuclide Therapy.

Peptide receptor radiotherapy using 177Lu-labeled somatostatin ligand analogs is a well-established treatment for neuroendocrine tumors, with 177Lu-DOTATATE having acquired marketing authorization in Europe and the United States. The investigation of the pharmacokinetics of these radiopharmaceuticals in vivo in humans is crucial for personalized treatment management and understanding of treatment effects. Such an investigation requires input data on the in vivo stability of the radiopharmaceuticals in blood and plasma. The work presented here is devoted to the investigation of the in vivo stability of 177Lu-DOTATATE in humans affected by neuroendocrine tumors. Methods: Blood samples of 6 patients undergoing 177Lu-DOTATATE were taken at 0.5, 4, 24, and 96 h after injection. Analysis of metabolic stability was performed using high-performance liquid chromatography. Results: A fast metabolism of the radiopharmaceutical was observed, with the fraction of intact 177Lu-DOTATATE in plasma decreasing rapidly to 23% ± 5% (mean ± SD) at 24 h and 1.7% ±0. 9% at 96 h after injection. Conclusion: The in vivo stability of 177Lu-DOTATATE is much lower than previously assumed, with the major part of radioactivity in plasma consisting of 177Lu-labeled metabolites already at 24 h after injection.

How to Achieve Approval of Radiolabeled-PSMA Diagnostic and Therapeutic Agents

The theranostic combination of 68Ga-labeled diagnostic agents with 90Y- or 177Lu-labeled therapeutic somatostatin ligands has been shown to be a remarkably accurate and effective way to characterize and treat neuroendocrine tumors. Food and Drug Administration approval of such agents is expected to

Constitutive Somatostatin Receptor Subtype 2 Activity Attenuates GH Synthesis

Somatostatin signals predominantly through somatostatin receptor (SSTR) subtype 2 to attenuate GH release. However, the independent role of the receptor in regulating GH synthesis is unclear. Because we had previously demonstrated constitutive SSTR2 activity in mouse corticotrophs, we now analyzed GH regulation in rat pituitary somatotroph (GC) tumor cells, which express SSTR2 exclusively and are devoid of endogenous somatostatin ligand. We demonstrate that moderately stable SSTR2 overexpression (GpSSTR2(WT) cells) was associated with decreased GH promoter activity, GH mRNA, and hormone levels compared with those of control transfectants (GpCon cells). In contrast, levels of GH mRNA and peptide and GH promoter activity were unchanged in GpSSTR2(DRY) stable transfectants moderately expressing DRY motif mutated SSTR2 (R140A). GpSSTR(2DRY) did not exhibit an enhanced octreotide response as did GpSSTR2(WT) cells; however, both SSTR2(WT)-enhanced yellow fluorescent protein (eYFP) and SSTR2(DRY)-eYFP internalized on octreotide treatment. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, increased GH synthesis in wild-type GC cells and primary pituitary cultures. GpSSTR2(WT) cells induced GH synthesis more strongly on SAHA treatment, evident by both higher GH peptide and mRNA levels compared with the moderate but similar GH increase observed in GpCon and GpSSTR2(DRY) cells. In vivo SAHA also increased GH release from GpSSTR2(WT) but not from control xenografts. Endogenous rat GH promoter chromatin immunoprecipitation showed decreased baseline acetylation of the GH promoter with exacerbated acetylation after SAHA treatment in GpSSTR2(WT) compared with that of either GpSSTR(2DRY) or control cells, the latter 2 transfectants exhibiting similar GH promoter acetylation levels. In conclusion, modestly increased SSTR2 expression constitutively decreases GH synthesis, an effect partially mediated by GH promoter histone deacetylation.

Pituitary tumors

The aim of this article is to review current research in the treatment of pituitary tumors and summarize emerging medical, surgical and radiation-based therapies. Existing medical agents such as dopamine agonists and somatostatin ligand receptors are being used in novel combinations to achieve biochemical remission for pituitary tumors and reduce tumor size. Advancements have also been made in surgery to improve tumor localization by use of intraoperative imaging and to reduce morbidities, such as cerebrospinal fluid leak, associated with transsphenoidal surgery. Finally, as radiation therapy and stereotactic radiosurgery for pituitary tumors gains more widespread use, long-term data is becoming available demonstrating its durability. Although current therapeutics and treatment modalities have been in practice for several years, new applications of existing treatments continue to be developed and have potential to improve care for patients with pituitary tumors.

Null mutant mouse models of somatostatin and cortistatin, and their receptors

Somatostatin (somatotropin release inhibitory factor, SRIF) and the related cortistatin (CST) are multifunctional peptide molecules attributed with neurohormone, neurotransmitter/modulator, and autocrine/paracrine actions. The physiological responses of SRIF and CST are mediated by five widely distributed G protein-coupled receptors (sst1-5) which have been implicated in regulating numerous biological processes. Much of the information on the effects of somatostatin has been gained through pharmacological studies with analogs and antagonists. The possibility of targeted mutagenesis in the mouse has resulted, over the last 10 years, in the generation of mouse models which genetically lack somatostatin ligands or receptors. We will review here the mouse models generated, the studies undertaken with them, and what has been learned so far.

Somatostatin Receptor Type 5 Modulates Somatostatin Receptor Type 2 Regulation of Adrenocorticotropin Secretion

Somatostatin inhibits adrenocorticotropin (ACTH) secretion from pituitary tumor cells. To assess the contribution of somatostatin receptor subtype 5 (SST5) to somatostatin receptor subtype 2 (SST2) action in these cells, we assessed multipathway responses to novel highly monoreceptor-selective peptide agonists and multireceptor agonists, including octreotide and somatostatin-28. Octreotide and somatostatin-28 cell membrane binding affinities correlated with their respective SST2-selective peptide ligand. Although octreotide had similar inhibiting potency (picomolar) for cAMP accumulation and ACTH secretion as an SST2-selective agonist, somatostatin-28 exhibited a higher potency (femtomolar). Baseline spontaneous calcium oscillations assessed by fluorescent confocal microscopy revealed two distinct effects: SST2 activation reduced oscillations at femtomolar concentrations reflected by high inhibiting potency of averaged normalized oscillation amplitude, whereas SST5 activation induces brief oscillation pauses and increased oscillation amplitude. Octreotide exhibits an integrated effect of both receptors; however, somatostatin-28 exhibited a complex response with two separate inhibitory potencies. SST2 internalization was visualized with SST2-selective agonist at lower concentrations than for octreotide or somatostatin-28, whereas SST5 did not internalize. Using monoreceptor-selective peptide agonists, the results indicate that, in AtT-20 cells, SST5 regulates the dominant SST2 action, attenuating SST2 effects on intracellular calcium oscillation and internalization. This may explain superior somatostatin-28 potency and provides a rationale for somatostatin ligand design to treat ACTH-secreting pituitary tumors.

The novel somatostatin ligand (SOM230) regulates human and rat anterior pituitary hormone secretion.

Currently available somatostatin analogs predominantly bind to the somatostatin receptor subtype (SSTR)2 subtype, and control GH and IGF-I secretion in approximately 65% of patients with acromegaly, their efficacy relating to receptor density and subtype expression. SOM230 is a somatostatin ligand with high affinity to four SSTR subtypes. In primary cultures of rat pituicytes, SOM230 dose-dependently inhibited GH release (P = 0.002) with an IC50 of 1.2 nM. Ten nanomoles SOM230 inhibited GH and TSH release by 40 +/- 7% (P < 0.001) and 47 +/- 21% (P = 0.09), respectively. No effect of SOM230 was observed on prolactin (PRL) or LH release. In cultures of human fetal pituitary cells, SOM230 inhibited GH secretion by 42 +/- 9% (P = 0.002) but had no effect on TSH release. SOM230 inhibited GH release from GH-secreting adenoma cultures by 34 +/- 8% (P = 0.002), PRL by 35 +/- 4% from PRL-secreting adenomas (P = 0.01), and alpha-subunit secretion from nonfunctioning pituitary adenomas by 46 +/- 18% (P = 0.34). In contrast, octreotide inhibited GH, PRL, and alpha-subunit from the respective adenoma by 18 +/- 12 (P = 0.39), 22 +/- 4 (P = 0.04), and 20 +/- 10% (P = 0.34). In all culture systems, no significant difference in the inhibitory action of SOM230, octreotide, and somatostatin 14 on hormone release was observed. SOM230, similar to somatostatin, has high-affinity binding to SSTR1, 2, 3, and 5 and, in keeping with this, has an equivalent inhibitory effect on pituitary hormone secretion. As a consequence of its broader binding profile, SOM230 is likely to find clinical utility in treating tumors resistant to SSTR-2-preferential analogs.

Effect of somatostatin on nitric oxide production in human retinal pigment epithelium cell cultures.

To investigate the presence of somatostatin and its receptors (sst(1-5) receptors) and their possible involvement in the regulation of nitric oxide (NO) production in human RPE cell cultures. Human RPE cells (D407) were used for all studies performed. Somatostatin levels were detected by radioimmunoassay, and RT-PCR and immunocytochemistry studies were performed to identify the somatostatin receptors (sst1-sst5). Radioligand binding assays were also performed examining the ability of certain somatostatin ligands (sst1, sst2, sst5) to compete for [125I]Tyr11 somatostatin binding. The presence of NO synthase in the cultures was assayed with NADPH-diaphorase cytochemistry, and RT-PCR, and NO levels were assessed by examining the production of its stable metabolites NO2- and NO3- (NOx-). SRIF was detected in a concentration of 0.56 +/- 0.13 picomoles/mg protein. sst1, sst2, and sst5 mRNAs were detected, yet only sst2B and sst5 immunoreactivity was observed in human RPE cell cultures. sst1- and sst5- but not sst2-selective ligands displaced the specific [125I]Tyr11 somatostatin binding to RPE cell membranes. NADPH-diaphorase stain and iNOS mRNA were detected. SRIF and the sst2-selective analogue MK678 increased the levels of NOx- in a concentration-dependent manner. This increase was blocked by the sst2 antagonist CYN-154806 (Ac-4NO2-Phe-c(dCys-Tyr-dTrp-Lys-Thr-Cys)-dTyr-NH2). These results demonstrate the presence of somatostatin, and its receptors sst1, sst2B, and sst5 in human RPE cells and suggest an autocrine or paracrine role for somatostatin. Somatostatin's ability to regulate NO production, by activating sst2 receptors, provides a functional role of somatostatin in the RPE.

Somatostatin Receptor Subtype 2 Gene Therapy Inhibits Pancreatic Cancer in Vitro

Background. Most human pancreatic adenocarcinoma cells do not express somatostatin receptors and somatostatin does not inhibit the growth of these cancers. We have demonstrated previously that somatostatin inhibits the growth of pancreatic cancers expressing somatostatin receptor subtype 2 (SSR2) but not receptor-negative cancers. SSR2 expression may be an important tumor suppressor pathway that is lost in human pancreatic cancer. We hypothesized that SSR2 gene transfer would restore the growth inhibitory response of human pancreatic cancer to somatostatin.Methods. We created adenoviral constructs containing the SSR2 or Lac-Z gene and transfected somatostatin receptor-negative human pancreatic cancer cells (Panc-1). Presence of functional cell surface SSR2 protein was assessed by whole-cell competitive binding assays. Parental cells, Lac-Z-transfected, and SSR2-transfected cells were cultured in the presence and absence of somatostatin. The rate of cell growth was determined by direct cell counting using a hemacytometer (n = 8 wells/group). Cells were analyzed for expression of tumor suppressor proteins by Western blot.Results. Panc-1 cells transfected with the SSR2 transgene demonstrated high-affinity specific binding of 125I-somatostatin at physiologic concentrations. Expression of somatostatin receptors caused 60% inhibition of cell growth compared with the Lac-Z virus-treated controls (P < 0.05 by Kruskal–Wallis/Bonferroni). There was no additional inhibition of cell proliferation with exogenous somatostatin. Furthermore, addition of somatostatin ligand antibody did not diminish the effect of SSR2 expression on cell proliferation. Western blot analysis revealed an upregulation of the cyclin-dependent kinase inhibitor p27 in the SSR2-transfected cells.Conclusions. Expression of SSR2 by human pancreatic cancer causes significant slowing of cell division by a mechanism independent of somatostatin. The mechanism may involve upregulation of known tumor suppressor proteins. Restoration of SSR2 gene expression deserves further study as a potential gene therapy strategy in human pancreatic cancer.

Molecular cloning of a novel G protein-coupled receptor related to the opiate receptor family.

A cDNA clone encoding a novel G protein-linked receptor was isolated from a rat cerebral cortex cDNA library using a polymerase chain reaction-amplified cDNA fragment as a probe. This 2.4-kb clone encodes a 367 amino acid protein with seven putative transmembrane spanning domains. The protein is highly homologous to the cloned micro, delta, and kappa opioid receptors and shares with them structural features such as three glycosylation sites in the amino terminus, a cyclic AMP-dependent kinase phosphorylation site in the third cytoplasmic loop, an aspartic acid residue in the second transmembrane domain, and a palmitoylation site on the intracellular carboxy terminus. The receptor is also homologous with members of the somatostatin receptor family, yet it binds neither opiate nor somatostatin ligands. Northern blot analysis reveals two transcripts of 3.2 and 7.6 kb that are predominantly expressed in the cerebral cortex and hypothalamus. In situ hybridization analysis also shows a high abundance of mRNA in the cerebral cortex, hippocampus, amygdala, hypothalamus, thalamus, and dorsal raphe nuclei. It is suggested that the endogenous ligand for this receptor may represent a novel neuropeptide that may be closely related to the opiate peptide family.

Somatostatin-binding sites on rat telencephalic astrocytes

Using a somatostatin-gold conjugate as ligand, high-affinity binding sites for this neuropeptide were demonstrated at three levels: (i) cultured astrocytes from rat cortex, (ii) hippocampal slice cultures, and (iii) frozen tissue sections of rat telencephalon. The conjugate proved as active as the native peptide in competing for the binding sites. Light-microscopic visualization of bound ligand was achieved by silver intensification of the colloidal gold. This method is faster and yields superior resolution compared with autoradiography. Cultured astrocytes from cortex and hippocampus could be labeled by the ligand. At the light- and electron-microscopic level, astrocytes could be double-labeled by the somatostatin-gold conjugate and immunostaining for glial fibrillary acidic protein (GFAP). In hippocampal slice cultures, the conjugate did not penetrate into the neuropil because of a covering glial layer. However, a portion of this completely GFAP-positive covering glia reacted with the somatostatin ligand. In frozen brain sections, apart from delicate punctate structures, two types of labeled glia cells were seen: single stellate astrocytes and perivascular glia cells.